In the biotechnology field today, one of the most widely used separation methods is chromatography. The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions i.e. partitions many times between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
The chromatographic methods suggested up to date are based on different modes of interaction with a target. Thus, for example, in ion-exchange chromatography, the functional groups are permanently bonded ionic groups with their counter ions of opposite charge, while in hydrophobic interaction chromatography (HIC), the interaction between the stationary phase and the component to be separated is based on hydrophobic. Other chromatographic separation principles are well known to the skilled person.
The stationary phase, also known as the separation matrix, comprises a support, which is commonly a plurality of essentially spherical particles, and ligands coupled to the support. In most separation matrices, the support is porous to allow a larger amount of ligand and consequently more bound target compound in each particle.
U.S. Pat. No. 6,428,707 (Amersham Biosciences AB) relates to a process for the separation of a substance by adsorption to non-packed beds containing beads which exhibit ligands with affinity to the substance. The disclosed process is stated to improve total yields in adsorption processes on fluidized beds; improve productivity on fluidized beds and provide matrices that have improved breakthrough capacity in fluidized beds. This was achieved according to the inventors by utilizing beads in which the ligand is linked to the base matrix of the beads via an extender. The positive effect noted in this patent and caused by the extender is according to the patent believed to reside in the fact that it will provide the inner surfaces (pore surfaces) and/or outer surfaces of the beads with a flexible polymer layer that is permeable to macromolecules and other molecules allowed to pass the bed, causing an increase in the effective interacting volume as well as in the steric availability of the ligands which in turn increases the mass transfer rate as well as the total capacity.
EP 1 764 152 (Millipore Corporation) relates to asymmetric porous adsorptive beads, which comprise first and second regions. At least one characteristic differentiates the first from the second region, which characteristic may be pores size distribution, ligand density, ligand type, ligand mixture, media material, and percent agarose.
U.S. Pat. No. 6,572,766 (Bergstrom et al) relates to a matrix including a core showing a system of micropores and a surface in which the micropore system has openings. According to this patent, it was possible to achieve co-operation of different separation principles on the same chromatographic medium and in this way reduce the number of necessary separation steps in a purification process. More specifically, this was achieved by coating the surface with a polymer having such a large molecular weight that it cannot penetrate into the micropores. Interesting micropores are in several cases smaller than 1 μm, but can also be larger, depending on the intended use of the finished matrix. The micropores correspond in many cases to diffusion pores.
U.S. Pat. No. 6,426,315 (Bergstrom et al) relates to process for preparing multifunctional porous matrices, and more specifically, to a process of introducing layered functionalities by introducing a desired functionality in one or more well defined layers in the matrix. This can be obtained by contacting a matrix with a functional deficiency of reagent I, and choosing conditions and reagent I so that the reaction between reagent I and the groups A is more rapid than diffusion of reagent I in the matrix. In many embodiments of the matrices of the invention, the substitution degree of a ligand in the surface layer is zero or close to zero, while at the same time the same ligand is present in an inner layer. Also the reversed can be true. According to this patent, ligands are typically coupled to the matrix via a bridge which can be of varying structure according to known techniques. The bridge structure can be polymeric, e.g. a hydrophilic or a hydrophobic polymer. Common bridge names have been “tentacles”, “extender”, “fluff”, “linker”, “spacer” etc. Examples of hydrophilic polymer bridges are polysaccharides, such as dextran, and other water soluble polyhydroxy polymers. The requirements concerning the porosity (exclusion limit) of the separation matrices are primarily determined by mole weight and shape of the compounds which are to be separated. For the invention, it is also important that the porosity shall permit transport within the matrix of reagent I and often also of compound B.
U.S. Pat. No. 4,927,879 (Pidgeon) relates to a method for solid phase membrane mimetics, and more specifically to a stationary phase chromatographic support material designed to mimic the structure of biological cell membranes. The membrane mimetic structure presents a hydrophilic outer portion and a hydrophobic inner portion and is covalently bound to a surface having reactive functional groups. Phospholipids having reactive functional groups on their hydrophobic portion can be used to form the artificial membrane structure. The disclosed support may be used to allow separation of a wide variety of peptides/proteins using an aqueous mobile phase without the added protein-denaturing solvents commonly used in reversed chromatography (RPC) systems.
Despite the technologies disclosed in the above-discussed prior art, there is still a need in this field of novel methods, preferably suitable for large-scale purification of biomolecules.